RNA Interference
RNA interference (RNAi) is a process by which a double-stranded RNA (dsRNA) selectively inactivates homologous mRNA transcripts by triggering specific degradation of homologous RNAs in the cell. RNAi is more potent than anti-sense technology, giving effective knockdown of gene expression with as little as 1-3 molecules of duplex RNA per cell. Furthermore, inhibition of gene expression can migrate from cell to cell and may even be passed from one generation of cells to another.
Traditionally, RNAi has required long pieces (200-800 base pairs) of dsRNA to be effective. This is impractical for therapeutic uses due to the sensitivity of long RNA molecules to cleavage by RNases found in the plasma and intracellularly. In addition, long pieces of dsRNA have been reported to induce panic responses in eukaryotic cells, which include nonspecific inhibition of gene transcription, but also production of interferon-α.
When long dsRNA duplexes enter the cytoplasm, an RNase III type enzymatic activity cleaves the duplex into smaller, 21-23 base-pairs molecules, termed small interfering RNA (siRNA). Short RNA duplexes are active in silencing gene expression but do not trigger nonspecific panic responses when less than 30 nucleotides in length. Moreover, siRNAs can be administered directly to a cell or organism to silence gene expression, thereby obviating the need to use long dsRNA or less effective single-strand anti-sense RNA, ribozymes, or the like.
siRNA has been found effective for inhibiting expression of a variety of genes in mammalian cells in vitro and in vivo. siRNA technology provides an appealing approach for selectively inhibiting gene expression in clinical and therapeutic settings due to many advantages over conventional gene and antibody blocking approaches, including: (1) potent inhibitory efficacy; (2) specificity—even a single nucleotide mismatch can be distinguished; (3) inhibitory effects that can be passed to daughter cells for multiple generations; (4) high in vitro transfection efficiency; (5) practicality for in vivo use due to short sequence length, low effective concentrations and lack of neutralizing antibody production; (6) availability of tissue- and cell-specific targeting (e.g. via inducible or promoter-specific vectors, ligand-directed liposomes or antibody-conjugated liposomes); and (7) possibility of simultaneously silencing multiple genes or multiple exons in a single gene.
Nevertheless, siRNA approaches are not without limitations. Not all 21-23 nucleotide regions in a gene can be effectively targeted. Certain positions within the siRNA appear to be more important than others for effectively inhibiting gene expression. Furthermore, “off-target” effects (i.e., silencing expression of unintended genes) can be seen with as short as a 6-7 nucleotide match between the siRNA and an off-target gene, in some cases leading to non-specific, toxic effects. Although progress is being made toward understanding the importance of these factors and devising formulas and algorithms for siRNA design, the selection of effective siRNAs remains at least in part an empirical exercise.
The Brother of the Regulator of Imprinted Sites (BORIS)
The BORIS gene encodes a germ line, testis- and cancer-specific, paralog of the CTCF (CCCTC-binding factor; GenBank Accession No.: NM—006565), and is an epigentically-acting transcription factor that represses the tumor inhibitor functions of CTCF. Thus, BORIS is also referred to as CTCFL for CTCF-like. BORIS contains a central DNA-binding domain that is nearly identical to CTCF, but differs in N and C termini amino acid sequence, thereby suggesting that BORIS could play a role of interfering with CTCF-driven regulatory pathways if it is abnormally expressed in somatic cells (Klenova et al., Semin. Cancer Biol. (2002) 12:399-414; Loukinov et al., Proc. Natl. Acad. Sci. USA (2002) 99:6806-11).
Abnormal activation of BORIS has been observed in all human primary tumors and cancer cell lines tested, including breast, lung, skin, bone, brain, colon, prostate, pancreas, mast cell, ovarian and uterine cancers, with increased expression associated with advanced stage of disease (see e.g., Ulaner et al., Hum. Mol. Genet. (2003)12:535-49; Vatolin et al., Cancer Res (2005) 65:7751-62; Hong et al., Cancer Res (2005) 65:7763-74; and Loukinov et al, J. Cell. Biochem. (2006) 98:1037-43; D'Arcy et al, Br. J. Cancer (2008) 98:571-9). BORIS induces de-repression of many genes associated with malignancy (Vatolin et al., Cancer Res (2005) 65:7751-62; Hong et al., Cancer Res (2005) 65:7763-74), and ectopic expression of BORIS in normal cells has been reported to result in classic features of cell-transformation (see Ghochikyan et al., J. Immunol. (2007) 178: 566-73).
BORIS reportedly competes with CTCF for shared DNA target sites and can tether epigenetic modifications to and around such sites, resulting in modulation of gene expression (see Vatolin et al., Cancer Res (2005) 65:7751-62; Hong et al., Cancer Res (2005) 65:7763-74; Ghochikyan et al., J. Immunol. (2007) 178: 566-73). BORIS can also bind methylated DNA target sites, while there is evidence that CTCF cannot (see Nguyen et al., Cancer Res (2008) 68:5546-51). Therefore, BORIS can be classified as a unique cancer-testis gene with cell-transforming activity that is most likely mediated by competition with somatic tumor suppressor CTCF through epigenetic modifications (Vatolin et al., Cancer Res (2005) 65:7751-62).
Previous studies have demonstrated the potential of BORIS as a target for anti-cancer therapeutics. Protein-based, but not DNA-based, BORIS vaccine induced a significant level of antibody production in immunized animals, leading to breast cancer regression. Interestingly, potent anticancer CD8−-cytotoxic lymphocytes were generated after immunization with a DNA-based, but not protein-based, BORIS vaccine. (Ghochikyan et al., J. Immunol. (2007) 178: 566-73). However, the applicability of immunological approaches to cancer treatment is subject to limitations, including a) tumor suppression of the host immune system through active production of soluble and membrane bound factors; b) ability of tumor cells to lose expression of antigen processing machinery; and c) possibility of a deficit in the immunological repertoire of cancer patients caused by down regulation of TCR zeta chain expression.
BORIS is a particularly appealing target for cancer therapy for several reasons. First, the widespread distribution of BORIS in different types of cancer cells coupled with the general concept that while non-malignant cells do not require activated oncogenes for survival, the suppression of an activated oncogene in a cancer cell often leads to apoptosis, suggests that therapies targeting BORIS may be effective for selective killing of a large number of cancer cell types. Thus, a single approach to cancer therapy may be applicable to many forms of cancer. Furthermore, BORIS is limited to testes and cancer cell types, and is not found in the vast majority of normal cell types. Therapies directed at BORIS are expected to have fewer side effects than others that target molecules or mechanisms present in normal cells, particularly in women where BORIS is not found in normal tissues.
Thus, there is an interest in developing anti-BORIS therapies for cancer, particularly in malignancies of the female reproductive system such as breast and ovarian cancer.